This invention relates to an improved process for transferring biological materials such as nucleic acid into the cytoplasm of living cells. With the rapid advancement of recombinant DNA technology, there is a wide-ranging need for biologists to transfer biologic substances from one cell to another, and to transfer synthetic biological material into living cells to exert their activity therein. Such materials can include biological stains, proteins (antibodies or enzymes), and, most commonly, nucleic acids genetic material (either RNA or DNA). Most of the techniques used are painstakingly slow and use methods which transport materials into, at most, only a few cells at a time. More recently, there has been developed a particle bombardment process which utilizes a particle gun, as described in Sanford, et al., 1987, "Delivery of Substances Into Cells and Tissues Using A Particle Bombardment Process," Journal of Particle Science and Technology 5:27-37, the disclosures of which are hereby incorporated herein by reference.
An earlier invention of one of the joint inventors, Dwight Tomes, relates to an improved particle gun which uses a gun having a rifled barrel. The disclosure of particle gun bombardment and the method of transport of biological materials such as DNA into living cells as described in Tomes, IMPROVED PARTICLE GUN, filed May 12, 1989, Ser. No. 07/351,075, now abandoned, is incorporated herein by reference.
The effectiveness of particle transport is, of course, measured by the ability of living cells into which the transported particles have been inserted to pick up and express the biological material. This, of course, depends upon a wide variety of conditions. The less the expression, the less successful the transport. Correspondingly, the more successful the expression of the living cells i.e. the extent that they pick up and express the transported biological material, the better the nucleic acid insertion experiments.
In the particle gun technique the biological material (DNA for example) is mixed with the carrier. The carrier generally is comprised of a substantially inert metal in the form of small beads which function as microprojectiles. Generally, the microprojectiles have a diameter within the range of about 1 micron to about 4 microns. These beads can be made from tungsten, palladium, platinum or gold or an alloy thereof. Tungsten is preferred for reasons of economics. However, tungsten generally does not give as good or as successful transport and expression as does gold unless used with the parent pre-treatment process. Beads can generally range from about 1 micron to about 1.5 microns in diameter.
In the most preferred process, the beads are mixed with a small amount of biological material such as DNA or RNA. This is mixed with calcium chloride and a certain amount of polyamine is added.
Generally the ranges of each of these ingredients should be as follows:
Twenty-five .mu.l of tungsten particles at a concentration of 15-400 mg in 2 ml sterile water are placed in a sterile 10 ml centrifuge tube and agitated to suspend the beads. The preferred amount of beads is 375 mg/2 ml. Twenty-five .mu.l of the suspended tungsten beads are placed in an Eppendorf tube and DNA is added at a concentration of 1 .mu.g/.mu.l with the amount varying between 1 .mu.land 20 .mu.l, the preferred amount being 10 .mu.l. A calcium chloride solution of 25 .mu.l and having a concentration between 1.0-4.0M, preferably 2.5M is mixed with the DNA/bead mixture.
While addition of spermidine to the biological material/microprojectile combination has been previously employed, it has now been more broadly discovered that addition of a variety of polyamines to mixtures of tungsten beads and DNA or RNA in preparing microprojectiles significantly improve rates of transformation. While not intending to be limited by theory, it is believed that the polyamine improves delivery of biological material to the cells in this process by improving adherence of the materials to the microprojectile beads. Suitable polyamines have been found to include, for example, spermine, spermidine, caldine, thermine, and the like. The preferred polyamine, spermine, has been found to be superior to the previously disclosed spermidine additive. Accordingly, at this point a polyamine, preferably spermine, in an amount of 10 .mu.l and a concentration between 0.05M and 0.5M, preferably at 0.1M is added followed by finger vortexing. This mixture is allowed to stand for 10 minutes prior to centrifugation for one to two minutes at 9,000 rpm. Centrifugation may be used but is not required. The microprojectile mixture forms a pellet at the bottom of the Eppendorf tube. Before use, supernatant is withdrawn from the tube to provide a final volume of 30 .mu.l.
The DNA/bead mixture is sonicated briefly to suspend the microprojectiles prior to use. The suspended microprojectiles carrying the biological material are transferred to the forward end of the macroprojectile by micropipette in aliquots of 1-5 .mu.l, with 1.5 .mu.l preferred.
Among the difficulties which have been encountered in the past in using microprojectiles is that large masses of DNA would clump onto the particles which make it harder to separate and also make it more likely that the particles will kill the cells during bombardment.
As can be seen, there is therefore a continuing need to develop microprojectile bombardment processes for improved transformation of biological materials into living cells by a process which will not kill the cells during bombardment, by a process which can utilize inexpensive tungsten beads, and by a process can use such lesser expensive beads and still achieve a highly successful transformation and expression. This invention has as its primary objective the fulfillment of this need.